Signals emanating from midline notochord and floor plate are thought to be important for patterning the vertebrate neural tube. l propose to use zebrafish cyclops (cyc);floating head (flh) double mutant embryos that lack both floor plate and notochord as a model system for studying how the central nervous system (CNS) develops in the absence of signals provided by midline structures. For example, cyc- ;flh- embryos also lack some primary and all secondary motoneurons. To identify genes that are involved in mediating midline signals, I will use a subtractive hybridization approach to isolate genes that are not expressed in cyc;flh mutant embryos, and thus may be involved in differentiation of notochord, floor plate and motoneurons. Isolated genes will be used as probes for in situ hybridization to wild type embryos to determine in which cells and when in development they are normally expressed. The subtractive hybridization approach should identify: 1. genes that are direct downstream targets of cyc or flh, 2. genes encoding signaling molecules or their downstream targets, 3. genes important for the differentiation of motoneurons and for axonal guidance by the floor plate, and 4. genes encoding other floor plate and/or notochord-specific functions. Insight into how correct patterning of the vertebrate CNS is accomplished will provide a framework for addressing questions of neuronal organization and function and the basis of neural disorders.